EWS-FLI1 and RNA Pol II coimmunoprecipitate in crosslinked protein granules. ELISA assays using an antibody to FLI1 measured interactions with FLI1-fusion proteins recovered by co-IP assays from crosslinked cells. The IP of EWSR1 (B-1) or RNA Pol II (CTD4H8) recovered V5-EWS-FLI1 (A) but not YS37 (B) protein expressed in HEK293T/17 cells by plasmid transfections (n = 3 each). (C) Crosslinked IP assays of EWSR1 and RNA Pol II also recovered endogenous EWS-FLI1 from A673 cells (n = 4). (D) Transmission electron microscopy detected protein particles recovered from crosslinked HEK293T/17 cells by IP of EWS-FLI1 (anti-FLI1 antibody, left) and RNA Pol II (CTD4H8, left), but not of YS37 (anti-FLI1 antibody, center). Scale bar inset represents 50 nm. Cumulative plots show diameters for particles imaged in IP samples for EWS-FLI1 (n = 24) or RNA Pol II (n = 77). All error bars represent standard error about the mean. Student's t-test was calculated assuming equal variances: (**) P < 0.01; (*) P < 0.05; n.s., not significant (P > 0.05).
